1. Field of the Invention
The present invention relates to a precursor of fluorescence-labeled probe, a fluorescence-labeled probe, a fluorescence-labeled plasmid, a DNA strand labeled by said fluorescence-labeled probe, and a method of preparing said fluorescence-labeled plasmid.
2. Description of Related Art
In the fields of gene therapies and genetic researches, it is very important to estimate die amount of gene introduced into a cell and to observe intracellular localization of the gene. To attain such a purpose, it is very important to visualize a DNA by labeling die DNA, using a fluorescence-labeled probe. Concerning a linear DNA strand, a method of labeling 5xe2x80x2-end of the linear DNA strand has been known as a method for producing a fluorescence-labeled probe to be used for the purpose described above. However, regarding a circular DNA strand, above described method can not be applied, as there is no 5xe2x80x2-end in the circular DNA strand. The method applied for the circular DNA strand typically utilizes an optical reaction. The method comprises the steps of introducing a fluorescence-labeled probe containing an azide group into the circular DNA strand and cleaving the circular DNA strand at aldehyde group of ribose in the DNA by irradiation of UV rays.
The conventional method using optical reaction described above requires strict reaction conditions, so that the method might be harmful for die function of the target gene. Therefore, a novel fluorescence-labeled probe capable of labeling a circular DNA strand, such as a DNA plasmid generally used in the gene therapy, achieved by a simple method under a mild condition have been desired.
Therefore, the inventors have produced a precursor compound of a novel fluorescence-labeled probe for DNA by covalent binding of a fluorescence substance, such as fluorescein isothiocyanate (FITC), wherein a conjugating group is bounded, with an aminoalkylated phenylamine. Then, amino group of phenylamine contained in said precursor was converted to diazonium salt to produce a novel fluorescence-labeled probe for DNA. Moreover, a fluorescence labeled plasmid was produced by conjugating said fluorescence-labeled probe and purine ring of guanine contained in the DNA plasmid through said diazonium group. By using the fluorescence-labeled probe for DNA according to this invention, a fluorescence-labeled DNA can be produced without deteriorating the functions of the gene. Moreover, the present invention enabled observation of gene uptake using a flowcytometer or a fluorescence microscope.
This invention is a precursor compound of a fluorescence-labeled probe for a DNA, comprising:
a residue of a fluorescence substance containing a hydrogen atom wherein said hydrogen atom is removed from said fluorescence substance;
a conjugating group that binds to said residue;
an alkylamino group having an alkyl group and amino group, said amino group being bonded to said conjugating group and said alkyl group being a linear or branched alkyl group having 1 to 16 carbon atoms; and
a phenylamine having phenyl group and amino group, said alkyl group being bonded to said phenyl group at the para-position of said phenyl group.
The structure of a typical precursor of DNA probe according to the present invention is illustrated in FIG. 1, comprising fluorescein isothiocyanate (FITC) coupled to aminophenyl ethylamine. In FIG. 1, residue of fluorescein, thioamide group, ethylamine group and aminophenyl group are indicated by (1), (2), (3) and (4), respectively. Here, said fluorescein, said thioamide group and said ethylamine group correspond to said fluorescence substance, said conjugating group and said alkylamino group described above, respectively.
The alkyl group may include a linear or branched alkyl group having 1 to 16 carbon atoms, preferably having 2 to 10 carbon atoms, more preferably in having 2 to 6 carbon atoms.
The conjugating group to be used in the present invention may include any functional group, so far as it is capable of reacting to amino group. The preferable conjugating group may include thioamide group, sulfonyl group and carbonyl group.
The fluorescence substance to be used in the present invention may include any substance so far as it is capable of emitting fluorescence and binding to said conjugating group. The preferable fluorescence substance may include fluoroscein, sulforhodamine, rhodamine, dansyl chloride and 7-chloro-4-nitrobenzoxyazole.
Furthermore, this invention is a fluorescence-labeled probe for a DNA, comprising:
a residue of a fluorescence substance containing a hydrogen atom wherein said hydrogen atom is removed from said fluorescence substance;
a conjugating group that binds to said residue; an alkylamino group having an alkyl group and amino group, said amino group being bonded to said conjugating group and said alkyl group being a linear or branched alkyl group having 1 to 16 carbon atoms; and
a phenyldiazonium group having phenyl group and diazonium group, said alkyl group being bonded to said phenyl group at the para-position of said phenyl group.
The structure of a typical DNA probe according to the present invention is illustrated in FIG. 2, comprising fluorescein isothiocyanate (FITC) coupled to ethylamine phenyl diazonium group. In FIG. 2, residue of fluorescein, thioamide group, ethylamine group and phenyl diazonium group are indicated by (5), (6), (7) and (8), respectively. Here, said fluorescein, said thioamide group and said ethylamine group correspond to said fluorescence substance, said conjugating group and said alkylamino group described above, respectively.
The alkyl group may include a linear or branched alkyl group having 1 to 16 carbon atoms, preferably having 2 to 10 carbon atoms, more preferably in having 2 to 6 carbon atoms.
Furthermore, this invention is a plasmid labeled by the fluorescence probe described above. The fluorescence labeled plasmid can be prepared by ligating said fluorescence probe with a plasmid through purine ring of guanine contained in DNA of said plasmid. The method for preparing such fluorescence labeled plasmid is included within the scope of the present invention.
The method for preparing the fluorescence labeled probe according to the present invention is shown in FIG. 3. The reference numerals of chemical compounds described below correspond to the reference numerals shown in FIG. 3. As shown in FIG. 3, an active group of a fluorescence substance (compound 1), such as FITC, and amino group of an aminoalkylated phenylamine derivative (compound 2), such as aminophenyl ethylamine, are reacted. Consequently, a fluorescence compound (compound 3), containing a phenylamino group at its terminal, can be obtained. A fluorescence diazo compound (compound 4) can be obtained by diazotizing amino group of said terminal phenyl amino group of the chemical compound 3 using NaNO2. When the chemical compound 4 is reacted with a DNA plasmid, the compound 4 binds to a purine ring of guanine contained in the DNA plasmid, as the terminal of chemical compound 4 being a diazonium: salt.
That is, this invention is a method for preparing a fluorescence-labeled plasmid, comprising the steps of:
preparing said fluorescence-labeled probe described above;
preparing a plasmid; and
binding said phenyldiazonium group contained in said fluorescence-labeled probe with said plasmid through purine ring of guanine, said guanine being contained in DNA of said plasmid.
Moreover, this invention is a method for preparing a fluorescence-labeled plasmid according to the method described above wherein said fluorescence substance is selected from a group consisting of fluoroscein, sulforhodamine, rhodamine, dansyl chloride and 7-chloro-4-nitrobenzoxyazole.
Furthermore, this invention is a method for preparing a fluorescence-labeled plasmid according to the method described above wherein said conjugating group is selected from a group consisting of thioamide group, sulfonyl group and carbonyl group.
The FITC-labeled plasmid can be obtained by the process described above. The method for preparing FITC-labeled plasmid according to the present invention has the advantage that the reaction conditions are mild. Therefore, a fluorescence label of the target gene can be performed without deteriorating the functions of the gene.